Project Summary Although the advent of combination antiretroviral therapy (cART) has dramatically increased the life expectancy of people living with HIV-1, paradoxically the prevalence of HIV-1-associated neurocognitive disorders (HAND) in people treated with cART, is on the rise. It is estimated that almost 30-60% of infected individuals on cART will go on to develop HAND, out of which at least 30% will have a co-morbidity of substance abuse. It is well recognized that HIV-1 infection and drug abuse go hand in hand, leading not only to compromised cART adherence but also to exacerbation of HAND. Drugs of abuse and HIV-1 viral proteins (transactivator of transcription - Tat & gp120) have been shown to accelerate HAND pathogenesis co- operatively. Furthermore, similar to HIV-1-positive subjects on cART, SIV-infected rhesus macaques on cART also exhibit increased glial activation, which was associated with dysregulation of various signature microRNAs (miRs). Emerging evidence also points to the role of drugs such as cocaine in mediating glial activation with global changes in miRs. In our preliminary studies, we have demonstrated that exposure of microglial cells to both Tat & cocaine resulted in increased activation of microglia (compared to cells exposed to either agent alone) and this, in turn, was associated with downregulation of brain-enriched miR-124 through an epigenetic pathway (miR-124 promoter DNA methylation) and a concomitant upregulation of MeCP2 (also p-MeCP2), and STAT3 - the predicted targets of miR-124. Furthermore, we also found that overexpression of miR-124 in microglia resulted in alleviation of Tat & cocaine-mediated activation of microglia. The premise of this proposal is that the effect of HIV-1 Tat and cocaine can lead to increased activation of microglia, thereby serving as a model for assessment of broader effects of these agents in the CNS. It is thus hypothesized that Tat & cocaine activate microglia via downregulation of miR-124 involving the unique MeCP2-STAT3 signaling axis. The hypothesis will be tested in two specific aims: AIM 1: Determine the epigenetic mechanism(s) underlying Tat &/or cocaine-mediated downregulation of miR-124 and its association with microglial activation in vitro. AIM 2: Determine the effect(s) of miR-124 overexpression in blocking Tat &/or cocaine-mediated induction of Mg activation in vitro. The findings from this study will set the stage for our long- term future goals (not a part of this study), of validating the cell culture findings in appropriate animal models of HAND & cocaine abuse, and of harnessing the epigenetic modifications associated with Tat & cocaine-induced CNS inflammation as therapeutic targets of HAND.